Synthesis of 5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-one's aryl Schiff base derivatives and investigation of carbonic anhydrase and cholinesterase (AChE, BuChE) inhibitory properties

Article abstract of DOI:10.1016/j.bioorg.2019.02.045

Carbonic anhydrase enzymes (EC 4.2.1.1, CAs) are metalloenzyme families that catalyze the rapid conversion of H₂O and CO₂ to HCO₃ ^– and H⁺. CAs are found in different tissues where they participate in various significant biochemical processes such as ion transport, carbon dioxide respiration, ureagenesis, lipogenesis, bone resorption, electrolyte secretion, acid-base balance, and gluconeogenesis. In such processes, many CAs are significant therapeutic targets because of their inhibitory potentials especially in the treatment of some diseases such as edema, glaucoma, obesity, cancer, epilepsy, and osteoporosis. Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) inhibitors are also valuable compounds for different therapeutic applications including Alzheimer's disease. In this work, we report a fast and effective synthesis of 5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-one's aryl Schiff base derivatives and also their CA and cholinesterases inhibitory properties. Our findings showed that these Schiff base derivatives, with triazole ring, found as strong CA and cholinesterases inhibitors.

Full text of DOI:10.1016/j.bioorg.2019.02.045

Accepted Manuscript
Synthesis of 5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-one’s aryl Schiff base
derivatives and investigation of carbonic anhydrase and cholinesterase (AChE,
BuChE) inhibitory properties
Musa Özil, Halis T. Balaydın, Murat Şentürk
PII:
S0045-2068(18)31343-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.045
YBIOO 2820
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 November 2018
12 February 2019
21 February 2019
Please cite this article as: M. Özil, H.T. Balaydın, M. Şentürk, Synthesis of 5-methyl-2,4-dihydro-3H-1,2,4-
triazole-3-one’s aryl Schiff base derivatives and investigation of carbonic anhydrase and cholinesterase (AChE,
BuChE) inhibitory properties, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.02.045
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Synthesis of 5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-one’s aryl Schiff base derivatives
and investigation of carbonic anhydrase and cholinesterase (AChE, BuChE) inhibitory
properties
Musa Özil1, Halis T. Balaydın2, Murat Şentürk3
1Recep Tayyip Erdogan University, Faculty of Arts and Sciences, Department of Chemistry
53100, Rize, Turkey
2Recep Tayyip Erdogan University, Education Faculty, 53200, Rize, Turkey
3Agri Ibrahim Cecen University, Pharmacy Faculty, 04100 Agri, Turkey
Abstract
Carbonic anhydrase enzymes (EC 4.2.1.1, CAs) are metalloenzyme families that catalyze the
-
rapid conversion of H2O and CO2 to HCO3 and H+. CAs are found in different tissues where
they participate in various significant biochemical processes such as ion transport, carbon
dioxide respiration, ureagenesis, lipogenesis, bone resorption, electrolyte secretion, acid-base
balance, and gluconeogenesis. In such processes, many CAs are significant therapeutic targets
because of their inhibitory potentials especially in the treatment of some diseases such as
edema, glaucoma, obesity, cancer, epilepsy, and osteoporosis. Acetylcholinesterase (AChE)
and Butyrylcholinesterase (BuChE) inhibitors are also valuable compounds for different
therapeutic applications including Alzheimer’s disease. In this work, we report a fast and
effective synthesis of 5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-one’s aryl Schiff base
derivatives and also their CA and cholinesterases inhibitory properties. Our findings showed
that these Schiff base derivatives, with triazole ring, found as strong CA and cholinesterases
inhibitors.
Keywords: 1,2,4-Triazole; Schiff base; Benzohydrazide; Carbonic anhydrase; Cholinesterase;
Enzyme inhibition.
* Corresponding author: Dr. Musa Özil; Phone: +90-464 2236126;
E-mail: musa.ozil@erdogan.edu.tr

1. INTRODUCTION
Carbonic anhydrase isoenzymes (CAIs) are found in lots of tissues where they participate in
several important biological processes.[1-5] Moreover, many CAIs are target molecules to be
used in the treatment of diseases such as glaucoma, cancer, edema, obesity, epilepsy etc. [6-
12]
Relations between various human CA isozymes and various organic compounds
including different types of uracil, some salicylic acids, and some other derivatives have been
investigated.[13,14] A recent study examined the inhibitory effect of some sulfonamides on
mammalian carbonic anhydrase enzymes.[15] Their anti-inflammatory, antimutagenic,
antiviral, anti-carcinogenic, antibacterial or anticancer activities were also reported.[16,17]
Alzheimer’s disease (AD) is defined as a neurodegenerative condition described by
abnormal behavior, intellectual reduction, and it is one of the major public health problems,
especially due to the increasing elderly population in developed countries.[18,19] In spite of
the fact that AD pathogenesis has not been clarified yet, one of the most important theories
was the ‘‘cholinergic hypothesis”.[20] The defect in the levels of acetylcholine (ACh) and
butyrylcholine (BCh) was observed in the brains of patients with AD. The inhibition of AChE
and BuChE enzymes that hydrolyze ACh and BCh neurotransmitters has become thus a
treatment option of AD.[20] For this reason, many research groups have conducted
investigations of the inhibitory activity for these enzymes involved in AD pathogenesis.
AChE catalyzes the hydrolysis of ACh, which has an important role in cognition and memory.
Because of the loss of cholinergic neurons in AD patients, ACh depletion observations
constitute a strategy in treatment. Drugs such as donepezil, tacrine, galantamine, and
rivastigmine are AChE enzyme inhibitors, mainly ACh inhibits hydrolysis and increases the
amount of ACh.[21] While this treatment works in about half of the patients for several years,
curative therapy continues to be an unachieved goal.[21,22] These drugs interact with the
active site of the AChE: tacrine, without altering the structure of the enzyme (being a
reversible inhibitor), whereas rivastigmine changes it.[23,24] The carbamoyl group of
rivastigmine was found to covalently bound to AChE, with the rest of the drug in the
catalytic site and with its phenol functional group exposed to the solvent.[24-28]
In terms of their important potencies, especially in pharmaceutic treatment as drug candidates,
nitrogen-containing heterocycles liked Triazoles are widely synthesized and biologically

tested.[29-33] 1,2,4-triazole rings are pharmaceutically well-known scaffolds. Some drugs
having this ring have anticonvulsant,[34] analgesic,[34] antibacterial,[35,36] anti-
inflammatory,[37] anticancer,[38-40] antitumor,[41] antifungal,[42,43] antimicrobial,[44,45]
and antitubercular,[46] effects. Known 1,2,4-triazole containing drug examples are shown in
Figure 1. It is understood that this heterocyclic system behaves as an attractive scaffold and
promote chemical diversity for aimed purposes in medicinal chemistry.
N
N
N
N
N
N
N
H3C
N
O
O
N
N
N
HO
N
OH
N
N
NH2
HO
F
F
OH
CH3
F
F
F
Ribavirin
Fluconazole
Voriconazole
N
N
N
N
N
N
N
N
N
CH3
N
H3C
N
CH3
N
N
N
N
Cl
H3C
CH3
N
Vorozole
Anastrazole
Letrozole
Cl
Me2N
N
N
N
N
N
N
N
N
H
Rizatriptan
Estazolam
Figure 1. Drugs with the inclusion of 1,2,4-triazole ring.
In this researches in which organic syntheses are widely used, it is mostly preferred
microwave irradiation technique as a more useful method in the recent years[47,48] because
of their higher yields, easier work-up, better reaction rate, and so on. According to the eco-
friendly approach, this method has unique benefits.[49-50]
Within the scope of this study, we introduce a synthesis of triazole cycle, contained with
congeneric groups, such as esters, hydrazides and Schiff base at N-2 and N-4 nitrogen atoms.

The yields and reaction times of all reactions were compared between microwave and
conventional methods.
To discover some novel CA, AChE and BChE inhibitors, new types of Schiff base derivatives
were synthesized and evaluated in this study.
2. EXPERIMENTAL AND METHODS
2.1. Chemistry
2.1.1. General information
For melting point detection, it was used as a Büchi electric melting point appliance. The
infrared spectra were recorded on a Spectrum 100 FTIR Spectrometer (Perkin Elmer) using
1
potassium bromide pellets. The H and 13C NMR spectra were run at 400 and 100 MHz,
respectively on an Agilent Premium spectrometer in deuterated DMSO with TMS as interior
standard. The elemental microanalyses (C, H, and N) were executed on a Carlo-Erba 1106
CHN analyzer. The electrospray ionization mass spectra was verified on using Thermo
Scientific Quantum (TSQ) mass spectrometer. Microwave-assisted reactions were executed in
a monomode CEM-Discover device using at 300 W maximum power. The vial was chilled to
60°C via air-jet cooling, after the completion of the reaction.
2.1.2. Synthesis of compound 4.
The corresponding compound 3 (2.47 g, 10 mmol) and an equivalent amount of sodium (0.23
g, 10 mmol) were mixed in absolute ethanol (15 mL). Then, the mixture was irradiated with
microwaves at 75°C for 3 min. After the acidic proton is released, 1.66 mL bromoacetic acid
ethyl ester (15 mmol) was added to the mixture and irradiated for an additional 3 min at 75°C.
After the reaction was over (TLC, eluent Hexane - AcOEt, 1:3), the solvent was evaporated
under vacuum. Finally, to obtain the solid product as pure, it was recrystallized from water.
Yield 2.65 g (80%), mp 98–100 °C (lit. 98-100).[49]
2.1.3. Synthesis of compound 5.
Compound 4 (1.66 g, 5 mmol) and excess NH2NH2·H2O (100% concentration) (9.52 ml, 200
mmol) were dissolved in absolute EtOH (10 mL) and the mixture was irradiated with

microwaves at 60°C for 15 min. When the reaction was ended (TLC, MeOH) the reaction
content was cooled to room temperature (RT). The separated solid was filtered and
recrystallized twice from EtOH. Yield (1.38 g, 91%), mp 215–217°C (lit. 215-217).[49]
2.1.4. General process for the preparation of Schiff base derivatives 6a-f
Conventional Method: The equimolar quantities of compound 5 (1 mmol) and the
corresponding arom. aldehyde compound (1 mmol) were dissolved in 30 mL EtOH containing
glacial acetic acid in catalytic amount. The reaction mixture was refluxed for 12 h. After the
finalization of the reaction (by monitoring with TLC, ethylacetate: Hexane, 3:1), the reaction
content was cooled to RT until a solid appeared. The resulting solid was recrystallized two
times from EtOH to afford compounds 6a-f.
Microwave Method: The mixture of compound 5 (1 mmol) and the corresponding arom.
aldehyde (1 mmol) in EtOH (10 mL) containing catalytic amount of glacial acetic acid was
taken in a microwave vessel. After the microwave irradiation of the mixture in the closed
vessels with pressure control at 120 °C and 300 W for 5 min (hold time), the final procedures
(work-up and purification) were executed as explained overhead.
4-(1-{2-[2-Benzylidenehydrazino]-2-oxoethyl}-3-methyl-5-oxo-1,5-dihydro-4H-
1,2,4-triazol-4-yl)-N'-[phenylmethylene]benzohydrazide (6a): Yield: 0.37 g, (76 %)
(for conv. method), 0.41 g, (86 %), (for mw method), mp 256-258 °C; FTIR (KBr):
3236 (NH), 3059 (Ar-CH), 2942 (Alip-CH), 1715 (C=Otriazol), 1682, 1656
1
(C=Ohydrazide), 1592 (C=N). H NMR (400 MHz, DMSO-d6) δ: 2.18 (s, 3H, CH3), 4.92
and 4.50 (s, 2H, CH2, cis and trans conformers, cis/trans ratio 71/29), 7.42-7.73 (m,
12H, Ar-H), 8.01 (s, 1H, NCH), 8.06 (d, J=8 Hz, 2H, Ar-H), 8.46 and 8.24 (s, 1H,
N=CH, E/Z geometrical isomers, E/Z ratio 72/28), 11.71, and 11.67 (s, 1H, NH, cis and
trans conformers, cis/trans ratio 70/30), 11.97 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 12.8 (CH3), 46.7, and 47.4 (CH2, cis/trans), 127.1, 127.2, 127.4, 127.6
129.2, 129.3, 130.5, 130.6, 133.7, 134.3, 134.7, 136.2 (Ar.C), 142.9 (C=N), 144.7,
148.0, and 148.6 (N=CH, E/Z), 153.4, 162.8, and 163.5 (C=O, cis/trans), 168.4 (C=O).
Calcd for C26H23N7O3 %: C 64.85, H 4.81, N 20.36; found %: C 64.83, H 4.80, N
20.37; Mass spectrum, m/z (Irel, %): 482.1 [M+H]+ (100).
4-(1-{2-[2-(4-Chlorobenzylidene)hydrazino]-2-oxoethyl}-3-methyl-5-oxo-1,5-dihydro-
4H-1,2,4-triazol-4-yl)-N'-[(4-chlorophenyl)methylene]benzohydrazide (6b): Yield: 0.41 g, (74
%) (for conv. method), 0.49 g, (89 %), (for mw method), mp 268-270 °C; FTIR (KBr): 3192

(NH), 3060 (Ar-CH), 2941 (Alip-CH), 1723 (C=Otriazol), 1675, 1651 (C=Ohydrazide), 1589
1
(C=N), 829 (C-Cl). H NMR (400 MHz, DMSO-d6) δ: 2.18 (s, 3H, CH3), 4.92 and 4.51 (s,
2H, CH2, cis and trans conformers, cis/trans ratio 70/30), 7.47-7.77 (m, 10H, Ar-H), 8.00 (s,
1H, NCH), 8.05 (d, J=8 Hz, 2H, Ar-H), 8.44 and 8.23 (s, 1H, N=CH, E/Z geometrical
isomers, E/Z ratio 72/28), 11.76, and 11.74 (s, 1H, NH, cis and trans conformers, cis/trans
ratio 68/32), 12.03 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 12.8 (CH3), 46.7, and
47.3 (CH2, cis/trans), 127.1, 127.2, 128.2, 129.1, 129.2, 129.3, 129.4, 133.3, 133.6, 134.9,
135.1, 136.2 (Ar.C), 143.0 (C=N), 143.4, 146.7, and 147.2 (N=CH, E/Z), 153.4, 162.8, and
163.6 (C=O, cis/trans), 168.5 (C=O). Calcd for C26H21Cl2N7O3 %: C 56.74, H 3.85, N 17.81;
found %: C 56.72, H 3.85, N 17.79; Mass spectrum, m/z (Irel, %): 550.2 [M+H]+ (100).
4-(1-{2-[2-(4-Bromobenzylidene)hydrazino]-2-oxoethyl}-3-methyl-5-oxo-1,5-dihydro-
4H-1,2,4-triazol-4-yl)-N'-[(4-bromophenyl)methylene]benzohydrazide (6c): Yield: 0.53 g, (83
%) (for conv. method), 0.60 g, (94 %), (for mw method), mp 253-255 °C; FTIR (KBr): 3214
(NH), 3066 (Ar-CH), 2988 (Alip-CH), 1717 (C=Otriazol), 1680, 1662 (C=Ohydrazide), 1591
1
(C=N), 685 (C-Br). H NMR (400 MHz, DMSO-d6) δ: 2.18 (s, 3H, CH3), 4.88 and 4.62 (s,
2H, CH2, cis and trans conformers, cis/trans ratio 74/26), 7.61-7.70 (m, 10H, Ar-H), 7.98 (s,
1H, NCH), 8.05 (d, J=8 Hz, 2H, Ar-H), 8.42 and 8.21 (s, 1H, N=CH, E/Z geometrical
isomers, E/Z ratio 73/27), 11.76, and 11.74 (s, 1H, NH, cis and trans conformers, cis/trans
ratio 70/30), 12.03 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 12.9 (CH3), 46.4, and
47.0 (CH2, cis/trans), 127.0, 127.4, 127.9, 128.7, 129.1, 129.4, 130.0, 133.1, 133.8, 134.6,
135.2, 137.4 (Ar.C), 143.6 (C=N), 144.4, 146.2, and 147.0 (N=CH, E/Z), 152.9, 163.1, and
163.4 (C=O, cis/trans), 167.9 (C=O). Calcd for C26H21Br2N7O3 %: C 48.85, H 3.31, N 25.00;
found %: C 48.82, H 3.31, N 25.02; Mass spectrum, m/z (Irel, %): 638.2, [M+H]+ (61), 640.1
[M+H+2]+ (100), 642.3 [M+H+4]+ (63).
4-(1-{2-[2-(3-Bromobenzylidene)hydrazino]-2-oxoethyl}-3-methyl-5-oxo-1,5-dihydro-
4H-1,2,4-triazol-4-yl)-N'-[(3-bromophenyl)methylene]benzohydrazide (6d): Yield: 0.48 g, (76
%) (for conv. method), 0.56 g, (88 %), (for mw method), mp 222-224 °C; FTIR (KBr): 3212
(NH), 3066 (Ar-CH), 2984 (Alip-CH), 1716 (C=Otriazol), 1681, 1668 (C=Ohydrazide), 1584
1
(C=N), 689 (C-Br). H NMR (400 MHz, DMSO-d6) δ: 2.17 (s, 3H, CH3), 4.84 and 4.60 (s,
2H, CH2, cis and trans conformers, cis/trans ratio 69/31), 7.54-7.72 (m, 10H, Ar-H), 7.92 (s,
1H, NCH), 8.02 (d, J=8 Hz, 2H, Ar-H), 8.39 and 8.19 (s, 1H, N=CH, E/Z geometrical

isomers, E/Z ratio 70/30), 11.74, and 11.70 (s, 1H, NH, cis and trans conformers, cis/trans
ratio 73/27), 11.97 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 12.8 (CH3), 47.3, and
47.9 (CH2, cis/trans), 126.8, 127.3, 127.7, 128.6, 129.2, 129.7, 130.1, 130.9, 131.6, 132.4,
133.1, 133.7, 134.5, 135.7, 137.0, 137.8 (Ar.C), 143.5 (C=N), 144.6, 145.9, and 146.9
(N=CH, E/Z), 153.1, 162.8, and 163.3 (C=O, cis/trans), 167.4 (C=O). Calcd for
C26H21Br2N7O3 %: C 48.85, H 3.31, N 25.00; found %: C 48.84, H 3.30, N 25.01; Mass
spectrum, m/z (Irel, %): 638.4, [M+H]+ (65), 640.3 [M+H+2]+ (100), 642.1 [M+H+4]+ (71).
4-(1-{2-[2-(4-Methoxybenzylidene)hydrazino]-2-oxoethyl}-3-methyl-5-oxo-1,5-
dihydro-4H-1,2,4-triazol-4-yl)-N'-[(4-methoxyphenyl)methylene]benzohydrazide (6e): Yield:
0.42 g, (77 %) (for conv. method), 0.49 g, (91 %), (for mw method), mp 244-246 °C; FTIR
(KBr): 3213 (NH), 3018 (Ar-CH), 2981 (Alip-CH), 1708 (C=Otriazol), 1683, 1661
(C=Ohydrazide), 1588 (C=N). 1H NMR (400 MHz, DMSO-d6) δ: 2.19 (s, 3H, CH3), 3.92 (s, 3H,
CH3), 3.94 (s, 3H, CH3), 4.82 and 4.60 (s, 2H, CH2, cis and trans conformers, cis/trans ratio
71/29), 6.74-7.82 (m, 12H, Ar-H), 8.01 (s, 1H, N=CH), 8.50 and 8.31 (s, 1H, N=CH, E/Z
geometrical isomers, E/Z ratio 72/28), 11.54, and 11.58 (s, 1H, NH, cis and trans conformers,
cis/trans ratio 70/30), 12.05 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 12.8 (CH3),
45.6, and 47.4 (CH2, cis/trans), 49.4, 49.6 (CH3), 127.0, 127.5, 128.2, 128.7, 129.4, 129.9,
131.0, 133.2, 134.1, 134.8, 143.2, 143.6 (Ar.C), 144.5 (C=N), 145.1, 145.8, and 146.2
(N=CH, E/Z), 153.1, 162.8, and 163.2 (C=O, cis/trans), 168.2 (C=O). Calcd for C28H27N7O5
%: C 62.10, H 5.03, N 18.10; found %: C 62.06, H 5.02, N 18.10; Mass spectrum, m/z (Irel,
%): 542.3 [M+H]+ (73), 564.0 [M+Na]+(100).[52]
4-(1-{2-[2-(4-Bromo-2-hydroxybenzylidene)hydrazino]-2-oxoethyl}-3-methyl-5-oxo-
1,5-dihydro-4H-1,2,4-triazol-4-yl)-N'-[(4-bromo-2-ydroxyphenyl)methylene]benzohydrazide
(6f): Yield: 0.47 g, (70 %) (for conv. method), 0.58 g, (86 %), (for mw method), mp 233-235
°C; FTIR (KBr): 3382 (OH), 3189 (NH), 3053 (Ar-CH), 2976 (Alip-CH), 1722 (C=Otriazol),
1672, 1668 (C=Ohydrazide), 1592 (C=N) 735 (C-Br). 1H NMR (400 MHz, DMSO-d6) δ: 2.14 (s,
3H, CH3), 4.82 and 4.73 (s, 2H, CH2, cis and trans conformers, cis/trans ratio 71/29), 7.12-
7.79 (m, 10H, Ar-H), 7.97 (s, 1H, N=CH), 8.21 and 8.10 (s, 1H, N=CH, E/Z geometrical
isomers, E/Z ratio 73/27), 9.54 (s, 1H, OH), 9.56 (s, 1H, OH), 11.70, and 11.63 (s, 1H, NH,
cis and trans conformers, cis/trans ratio 69/31), 11.94 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 12.7 (CH3), 47.0, and 47.6 (CH2, cis/trans), 113.4, 114.1, 120.5, 121.3, 123.7,
125.6, 126.2, 126.9, 127.1, 128.0, 131.8, 133.4, 134.6, 135.5, 149.6, 150.3 (Ar.C), 144.5

(C=N), 145.2, 146.4, and 146.9 (N=CH, E/Z), 154.1, 163.8, and 164.7 (C=O, cis/trans), 167.8
(C=O). Calcd for C26H21Br2N7O5 %: C 46.52, H 3.15, N 14.61; found %: C 46.53, H 3.15, N
14.60; Mass spectrum, m/z (Irel, %): 670.1, [M+H]+ (65), 672.1 [M+H+2]+ (100), 674.2
[M+H+4]+ (67).[52]
2.2. Purification of hCA isozymes from erythrocytes by affinity chromatography
hCA I and II isoenzymes were purified from fresh human erythrocytes as described in the
literature [6-8]
2.3. Esterase activity assay
hCA I and II isoenzyme activity assays were carried out according to the Verpoorte
method.[53] In this part of the study, the inhibitory effects of 6a-f derivatives were tested.
These derivatives were examined in triplicate at each concentration used. Different
concentrations were used for all inhibitors. In the absence of inhibitor, the control cuvette
activity was accepted as 100%. Activity (%) - [Inhibitor] graphs were plotted for each
inhibitor.
2. 4. AChE and BChE activity
Inhibitory activities of the compounds on AChE and BuChE enzymes were
determined by using the Ellman method.[54] Neostigmine was used as the reference drug in
this study. The IC50 values obtained for compounds 6a-f are summarized in Table 1.
The stock solutions of all the materials tested were prepared by dissolving in 1 mg /
mL of dimethylsulfoxide. It was then diluted to different concentrations using deionized
water. Six serial dilutions of these substances were measured to determine the inhibitory
activity of AChE and BChE enzymes [55,56]
3. RESULTS
3.1. Chemistry
The synthetic route was started with producing compound 1. In the first step, to obtain
compound 1, the acetonitrile with EtOH and HCl(g) were reacted according to the Pinner

method.[57] Later, the ethyl 2-(1-ethoxyethylidene)hydrazine carboxylate (2) was obtained
from compound 1 and the ethyl carbazate at 0-5 °C.[58] Compound 2 reacted with 4-
Aminobenzoic acid ethyl ester under solvent-free condition using with the microwave
technique to produce ethyl 4-(3-methyl-5 oxo-1,5-dihydro-4H-1,2,4-triazole-4-yl)benzoate
(3). In the next step, compound 3 was reacted with the Bromoacetic acid ethyl ester under
both the conventional and microwave irradiation methods to obtained ethyl-4-[1-(2-ethoxy-2-
oxoethyl)-3-methyl-5-oxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]benzoate (4). To achieve aimed
synchronous modification, compound 5, which has two identical functional groups, was
obtained from compound 4 with excess hydrazine hydrate as described in the literature.[47]
Hydrazide compounds can be considered as a versatile group leading to the formation of
different heterocycle systems such as 1,3,4-oxadiazole, 1,2,4-triazole, and 1,3,4-thiadiazole
derivatives. [49] On the other hand, these compounds are very ambitious derivatives to
reacted with aldehydes to obtained Schiff bases. In our earlier studies, Schiff bases derivatives
which containing with 1,2,4-triazole-5-one ring system were prepared from aromatic amines
and different aldehydes. [35,58] However, in this study to obtain the biologically active target
compounds 4-[1-(2-{2-[4-(substitute)benzylidene] hydrazino}-2-oxoethyl)-3-methyl-5-oxo-
1,5-dihydro-4H-1,2,4-triazol-4-yl]-N'-{[4-(substitute) phenyl]methylene}benzohydrazide (6a-
f), compound 5 (hydrazide derivative) was performed with diverse aromatic aldehydes in
concur with acetic acid as a catalyst. These reactions were in the moderate yield (70-83%)
using conventional heating, and in high yield (86-94%) by means of the microwave as an
energy source (Scheme 1). The reaction times also were shortened from 12 h to 5 min by
using the microwave technique. When compared with the conventional (thermal) heating
method, microwave irradiation heating put forwards more advantages such as simplicity in
processing, low cost, reduced pollution, reduced reaction time, and high yield.

Scheme 1. The synthetic route for the 1,2,4-triazole derivatives
3.2. Biological Studies
This study reported the synthesis of Schiff base derivatives (6a-f) and also determined the
inhibitory effects of the compounds 6a-f on human CA I and II isoenzymes and AChE and
BChE enzymes.
AChE and BChE enzymes are known to have very important roles in memory and cognition.
These cholinergic enzymes catalyze the hydrolysis of ACh, which leads to a decrease in
neural communication between nerve cells. This leads to the function of the brain and finally

to the AH. Therefore, the treatment of AH depends to some extent on the rebalancing of the
level of ACh.[25-28] In many pharmaceutical studies, cholinesterase inhibitors (ChEIs) have
been targeted to treat cognitive disorders. Because of its disadvantages such as AD drugs,
bioavailability, and gastrointestinal disorders, new ChEIs are being investigated continuously.
This study thus screened derivatives 6a-f for their inhibitory activity.
Here, we first report the inhibition effects of the newly synthesized 6a-f derivatives using the
esterase activity of human CA I and CA II isoenzymes. In Table 1, the inhibition values
determined by the esterase activity of 6a-f compounds for hCA I and hCA II isoenzymes are
shown below,[53] with 4-NPA (4-nitrophenylacetate):
Aryl
H3C
N
Comp.
Comp.
Aryl
Aryl
HN
C
O
N
N
N
O
6d
6e
6f
6a
6b
-C6H4(m-Br)
-C6H5
-C6H4(p-Cl)
-C6H4(p-Br)
-C6H4(p-OCH3)
-C6H3(2-OH, 4-Br)
HN
6c
O
6a-f
N
Aryl
Figure 2. Tested compounds’ molecular structures.
Table 1: hCA I, II, AChE and BChE inhibition data some compounds.
IC50 (µM)*
Inhibitor
hCA I
hCA II
AChE
BChE
0.1110
0.0769
0.1361
0.1401
0.0538
0.0774
0.9857
0.0780
0.0762
0.1243
0.1170
0.0514
0.0717
0.4894
0.0465
0.0574
0.0877
0.0966
0.0623
0.0577
-
0.0702
0.0557
0.1253
0.0937
-
6a
6b
6c
6d
6e
0.0486
-
6f
AZAa
-
-
0.136
0.084
Neostigmine
Mean from at least three determinations. Errors in the range of ± 3% of the reported value
(data not shown). a From Ref.[67]

(i) Against the hCA I isozyme, the compounds 6a-f behaved as good inhibitors, with
the values of IC50 in the range of 0.0538-0.1401 μM, similar to structurally related compounds
N-benzyl-2-(2-hydroxybenzoyl) hydrazine carbothioamide (IC50 of 0.834 M). In this study,
compounds 6b, 6e, and 6f show much better hCA I inhibition effect than 6a, 6c, and 6d. For
compounds 6a-f, it was found that many previous kinetic studies showed similar results to
phenolic compounds, sulphonamide derivatives, uracil derivatives, natural products, and
anions.[6-17,59-63]
(ii) In this study, it was determined that 6a-f derivatives showed better inhibitory
activity for h CA II isoenzyme compared to hCA I isoenzyme (Table 1). If we analyze the
structure-activity relationship for 6a-f compounds, The compounds 6c and 6d which
containing Br are lower effective than other compounds. The best hCA II inhibitor in the 6a-f
derivatives is the 6e containing the methoxy group in the para position in the structure, the
IC50 of this substance being determined as 0.0514 M. For hCA II isoenzyme, 6a, 6b, and 6f
yielded very close to each other in the range 0.0717 to 0.780 µM. As human CA I and II
isoenzymes exhibit poor esterase activity, IC50 values are mostly in micromolar range when
measured according to the esterase method for these enzymes.[63]
(iii) Derivative 6d (IC50 = 0.0966 μM) showed the weakest inhibitory against AChE.
Decreasing one para position chloride group on the aromatic ring showed an improvement of
the IC50 values obtained, 6a (0.0465 μM) with p-Cl group demonstrated a 1.23 fold decrease
of inhibition activity while 6b (0.0574 μM) with para position bromide group 6c showed a
1.89 fold decrease of inhibition activity compared to compound 6a. However, compound 6a
(0.0465 μM) possessing only the benzene group showed better inhibitory activity compared to
the other five molecules. The difference between the other tested compounds and 6a is that
this molecule is a more voluminous derivative. The fact that only the benzene group
containing derivative 6a has a stronger inhibitory capacity as a functional group may indicate
that its geometry interacts with the enzyme active site is more potent. According to these
results obtained for AChE enzyme and 6a-f derivatives, it can be indicated that the
electronegative groups in the molecule have a great effect on the inhibition value according to
the position. Larger research will be conducted to explain this situation. To find out the effect
of the benzene group on the inhibitory activity as a functional group, five other compounds
(6b-f) were compared with compound 6a. Adding a 2-hydroxyl and 4 bromide group (6f) to
just benzene group-containing compound (6a) the inhibitory activity decreased 1.24 fold, it
only suggests that the benzene group had a significant inhibitory effect on AChE enzyme

activity. In the structure, a chlorine-containing compound 6b (0.0574 µM) in the para position
appears to have an inhibitor value of 1.23 times less than the 6a molecule. In addition, in our
study, the values (0.0465-0.0966 µM) obtained for AChE enzyme are better when they are
shortened by the reference molecule, neostigmine (0.136 µM).
(iv) Derivative 6c (IC50 = 0.1253 μM) showed the weakest inhibitory effect against the
BChE enzyme. Decreasing one para position bromide group on the aromatic ring showed an
improvement of the IC50 values obtained, 6f (0.0486 μM) with 2-OH and 4-Br groups
demonstrated a 2.58 fold decrease of inhibition activity while 6c (0.1253 μM) with para
position bromide group 6c showed a 1.93 fold decrease of inhibition activity compared to
compound 6f. However, compound 6f (0.0486 μM) possessing 2 hydroxy and 4 bromide
group showed better inhibitory activity compared to the other five molecules. The difference
between the other tested compounds and 6f is that this molecule is a more voluminous
derivative. The fact that 2 hydroxy and 4 bromide groups containing derivative 6f has a
stronger inhibitory capacity as a functional group may indicate that its geometry interacts with
the enzyme active site is more potent. According to these results obtained for BChE enzyme
and 6a-f derivatives, it can be indicated that the electronegative groups in the molecule have a
great effect on the inhibition value according to the position. Larger research will be
conducted to explain this situation. To find out the effect of the benzene group on the
inhibitory activity as a functional group, five other compounds (6a-e) were compared with
compound 6f. In the structure, a chlorine-containing compound 6b (0.0557 µM) in the para
position appears to have an inhibitor value of 1.15 times less than the 6f molecule. In addition,
in our study, it was observed that the IC50 values (0.0486-0.1253 µM) obtained for BChE
enzyme were close or better when they were shortened by neostigmine (0.0840 µM) as the
reference molecule.
In many recent studies, it has been found that simple phenolic compounds show good
inhibition as sulfonamides, which are potent CA inhibitors.[16] This may provide a new class
of inhibitors that is advantageous for the treatment of patients with sulfonamide allergy.[13-
17] Sulphonamides are the best known CA inhibitors, but many studies in recent years
indicate that molecules containing different functional groups (especially those containing
electronegative atoms) may exhibit as good inhibition as sulfonamides.[6-15,59-63] In
addition, it is critical to investigate more potent CAI classes to identify substances that may
exhibit a different inhibition profile as compared to sulfonamides and to find different
applications for inhibitors of CA isoenzymes.

4. DISCUSSION
The molecular structures of newly obtained compounds were demonstrated in details on the
strength of different spectral analyses and all data are compatible with our proposed
structures. According to the FTIR results for all compounds, it is seen a C=N signal at about
1588 cm-1 and C=O signals at about 1685 cm-1 for hydrazide group and at about 1715 cm-1 for
triazole ring. On the other hand, the compounds 6a-f had no –NH2 signal because of the fact
that the imine constitution and new C=O signals were a shift from 1657 cm-1 to 1685 cm-1.
1
In the H-NMR spectral data, N-H signal has been shown at about 12.03 ppm and
1
11.74 ppm for compounds 6a-f. When H-NMR data of 6a-f have been checked, it was seen
that these compounds’ some protons have 2 sets of the signal at the different ppm. Arylidene-
hydrazide structure in the compounds which exist as E/Z geometrical isomer from C=N
double bond and cis/trans amide conformers at the CO-NH single bond. As seen in the essay
[65,66]
the compounds which have C=N double bond would rather E geometrical isomer in
DMSO-d6 and Z isomers can be preferred in less fewer solvents by an intramolecular
1
hydrogen bond. All H NMR spectra the -NH, -N=CH, and N-CH2 groups showed 2 sets of
signals because of cis/trans conformers. The -NH signal was observed as 2 sets due to
trans/cis amide conformers at about 12.03 ppm and about 11.74 ppm for compounds 6a-f. On
the other hand, 2 sets of signals at about 8.42 and about 8.20 ppm were interested into the -
N=CH bond of the E and Z geometrical isomers, respectively. As seen in the essay, the
downfield lines of the imine proton were attributed to the E isomer of the amide structure,
while the upfield lines were assigned to the Z isomer of compounds 6a-f. Thirdly, the N-CH2
signal was monitored again as 2 sets as a result of trans/cis amide conformers at about 4.55
ppm and at about 4.88 ppm for compounds 6a-f. The proportion has been calculated by using
13
1H-NMR and C-NMR data in each case. E/Z geometrical isomer and cis/trans conformer of
1
compounds 6a-f and selected H-NMR spectrum of compound 6a were given in Scheme 2
and Figure 3, respectively. Furthermore, compounds 6a-f gave elemental analysis data and
mass spectra coherent with the appointed structures.

O
O
N
H
N
Ar
R
N
H
R
N
H
Ar
H
trans, Z
trans, E
O
H
N
O
H
N
H
Ar
R
N
R
N
Ar
H
cis, E
cis, Z
Scheme 2. cis/trans isomers and E/Z geometrical isomers of compound 6a-f
Figure 3. 1H NMR Spectrum of compound 6a

The 6a-f compounds used in this study showed different inhibition effects for hCA I and hCA
II isoenzymes due to the presence of different functional groups (Cl, Br, OH, OCH3, and
Benzyl) found in their molecular structures. The results obtained in this study show that, in
addition to the sulfamates/sulfonamides known as potent inhibitors in CA inhibition, there are
possible new classes of CAIs. In fact, these synthesized compounds were determined using
the esterase method in which they were highly effective hCAI and hCA II inhibitors at low
micromolar concentrations. The data obtained in this study show that these synthesized
compounds can potentially be used to produce strong inhibitors that target AChE and BChE
enzymes, as well as other CA isoforms that have not yet been tested (in Figure 4 and 5).
Figure 4: CA inhibition with zinc binders such as sulphonamides (A) and phenol (B) and
respected compound 6e (C). Figures represent distances (in Å), as determined by X-ray
crystallographic techniques.[12,63] Hydrogen bonds are represented as dashed lines. All these
binding modes have been proven by means of X-ray crystallography on enzyme-inhibitor
adducts.[63]

Figure 5. A: the binding of acetylcholine with the active region of the hAChE enzyme (pdb:
4ey4) [19], B: the binding pattern of butyrylcholine with the active site of the hBChE enzyme
(pdb: 1p0i) [19], C: the estimated AChE binding form of 6a, D: the predicted BChE binding
pattern of 6f.
If the results of this study are discussed, the findings may lead to interesting
interpretations and may help to develop new and effective drugs to slow down or stop AD. In
this study, in addition to comparing these synthesized molecules with other compounds, it
will further expand targets for determining the structure-activity relationship of 6a-f
derivatives. These newly synthesized compounds can also be used as drug precursors or
building blocks in the preparation of more effective drug molecules.

Acknowledgments
The authors gratefully to the “Scientific and Technical Research Council of Turkey
(TÜBİTAK)” for financial support through Project 112T640.
Conflict of Interest
The authors declare no conflict of interest.
ORCID
Musa Özil http://orcid.org/0000-0002-1980-1364
Halis Türker Balaydın http://orcid.org/0000-0003-2823-4004
Murat Şentürk https://orcid.org/0000-0002-9638-2896
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Highlights:
 Synthesis of triazole derivatives by using microwave and conventional method
 Isomers and conformers studied of new molecules
 Carbonic anhydrase and cholinesterase inhibitory properties
 Identification of a new class of inhibitors
 Structure Activity Relationship established

Graphical Abstract
Synthesis of 5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-one’s aryl Schiff base derivatives and
investigation of carbonic anhydrase and cholinesterase (AChE, BuChE) inhibitory properties
Musa Özil, Halis T. Balaydın, Murat Şentürk
H
Aryl
N
O
N
C
N
IC50 (µM)
hCA II AChE
6a-f
Inhibitor
Aryl
-C6H5
hCA I
BChE
6a
6e
6f
-
-
0.0465
-
-
O
CH3
-C6H4(p-OCH3)
0.0538
-
0.0514
-
-
-
N
NH
N
N
Aryl
-C6H3(2-OH, 4-Br)
0.0486
O